Antibiotic resistant bacterial pathogens cause more than 2 million new infections and 23,000 deaths each year in the US,1 indicating the ineffectiveness of our existing arsenal of antibiotics. Even when antibiotics are effective at eradicating infection, most are inherently nonspecific for pathogens and have unintended conse- quences on beneficial microbiota.2 Bacterial glycoproteins are intriguing targets for the selective treatment of bacterial diseases, as they contain rare monosaccharides that are expressed on a small number of bacterial pathogens, are absent from human cells, and are frequently linked to pathogenesis.3-5 Despite the importance of bacterial glycoproteins as a virulence factor, the systematic study and inhibition of bacterial glycosylation enzymes remains challenging due to their utilization of rare deoxy amino sugars and their production of prod- ucts that are refractory to traditional glycan analysis. The deployment of chemical tools that facilitate the study and inhibition of bacterial glycoprotein biosynthesis is a crucial step toward inactivating pathogens based on their distinctive glycans. The objective of this application is to apply metabolic glycan labeling to study and per- turb glycoprotein biosynthesis in the priority pathogen Helicobacter pylori (Hp). The central hypothesis of the application is that azide-bearing monosaccharides, including rare bacterial monosaccharide analogs,13 will fa- cilitate the tracking of bacterial glycoprotein biosynthesis and the identification of pathway inhibitors. Our hy- pothesis has been formulated on the basis of strong preliminary data produced in my laboratory, including the application of azidosugars to identify more than one hundred candidate Hp glycoproteins, to discover the genes responsible for their biosynthesis, and to establish that Hp glycosylation mutants display a range of functional defects.14-16 The rationale for the proposed research is that new targets of therapeutic intervention will be revealed, resulting in innovative approaches to treat Hp infection. Though this work focuses on Hp as a test bed, the results of this program will have broad implications for the study and inhibition of glycoprotein bio- synthesis in diverse priority pathogens.17 Guided by strong preliminary data, this hypothesis will be tested by pursuing two specific aims: 1) Understand how Hp's glycoprotein biosynthetic pathway assembles glycans; and 2) Develop small molecule inhibitors of Hp's glycoprotein biosynthetic pathway. Under the first aim, analy- sis of glycans produced in a panel of glycosylation mutants versus wildtype strains will reveal how Hp glycosyl- ation enzymes act together to produce higher-order glycans. Under the second aim, substrate-based analogs of rare bacterial monosaccharides will be screened for their ability to terminate or divert glycan biosynthesis and cause functional defects. The proposed studies will uncover and harness glycosylation enzymes as antibi- otic targets, setting the stage to evaluate glycosylation inhibitors as a means to treat Hp infection. More broad- ly, this work will yield a novel approach to studying and inhibiting bacterial glycoprotein biosynthesis, opening the door to targeting myriad pathogenic bacteria with selectivity based on their distinctive glycans.